The quality of life for over 2 million Americans with celiac disease (CD) is significantly compromised because the only treatment is strict compliance to a gluten-free diet. Given the prevalence of gluten-containing staple foods, new therapeutic options that target the inappropriate immune response triggered by this common dietary staple must be developed. Thus, the overall goal of our project is to determine the inflammatory cell subset that drives pathogenic adaptive immune responses in CD and identify the activating receptor(s) that could be down-regulated by conditioning factors or blocked by antagonists to restore gluten tolerance in CD. The major genetic risk factor in CD is inheritance of HLA class II alleles, with 95% of patients expressing HLA- DQ2 and the others HLA-DQ8. However, it does not explain why the majority of HLA-DQ2+/8+ individuals (~35% of the general U.S. population) tolerate glutens. Compelling new studies indicate that additional factors related to innate immunity are fundamental to the pathogenesis of CD. An essential component appears to be uncontrolled expression of the cytokine interleukin (IL)-15. While the downstream effects of IL-15 are well recognized in CD, the receptor-ligand interactions that induce its production and that of other proinflammatory mediators remain unknown. Increased expression of toll-like receptor (TLR)4 has been detected in the intestine of treated and untreated CD patients compared to healthy individuals, suggesting the possibility that this pattern recognition receptor contributes to disease. Our preliminary data provide primary evidence that digested gliadin proteins from wheat gluten (PTG) stimulate TLR4 to mediate production of IL-1b, IL-15, IL-23 and TNF?. Based on these findings, we will test the hypothesis that interactions between digested gluten fragments and the TLR4 complex activate proinflammatory immune responses that perpetuate intestinal inflammation in CD. Aim 1 will (i) identify the cell types that express the TLR4 complex and IL-15 in the intestine of untreated CD patients and (ii) determine if this inflammatory cell subset is increased in the gut of untreated CD patients compared to treated CD patients and healthy individuals. Aim 2 will establish if the TLR4 complex is involved in the proinflammatory innate immune response of to PTG in HLA-DQ2+ individuals without and with CD. The proposed studies will identify the cell source(s) of TLR4 and IL-15 that are increased in active celiac lesions and determine if the TLR4 complex is directly involved in the inflammatory response of lamina propria mononuclear cells to PTG. This information will provide the underpinnings for an R01 application that will lead to new therapeutic strategies in CD. Thus, this grant will achieve a lasting impact on the field.